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研究生:譚皓仁
研究生(外文):Hao-Jen Tan
論文名稱:創傷弧菌低鹽適應與RpoS的關係
論文名稱(外文):Relationship between RpoS and the low salinity adaptation of Vibrio vulnificus
指導教授:黃顯宗黃顯宗引用關係
指導教授(外文):Hin-chung Wong
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:83
中文關鍵詞:創傷弧菌低鹽適應
外文關鍵詞:Vibrio vulnificuslow salinity adaptationRpoS
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創傷弧菌(Vibrio vulnificus)是一種生存在海洋環境中的革蘭氏陰性弧菌,生長於河流入海口沿海環境,可以透過傷口的感染或食用未煮熟的海鮮而造成感染,會造成原發性敗血症,嚴重時會導致死亡。不論在台灣或世界皆視為極為重要的食品中毒病原菌。在食品處理過程中,致病菌通常會遭受到許多環境壓力的影響,如高溫、低溫、酸、乾燥、滲透壓等,致病菌在非致死的壓力下通常會產生適應反應而增強對環境的抗性,也直接影響提高食品中毒的機率。
為了面對生長環境經常的改變,創傷弧菌必須有所機制可以抵抗環境變化所產生的壓力。目前研究已證實RpoS在細菌適應如飢餓、溫度、滲透壓、和氧化壓力等環境壓力中扮演極為重要的角色。本研究探討不同低鹽濃度對創傷弧菌的影響,並觀察經過非致死低鹽濃度適應的創傷弧菌對低鹽、熱、酸和膽鹽等壓力是否產生抗性,同時利用創傷弧菌rpoS突變株來探討低鹽適應與RpoS的關係。另外也觀測在適應前後創傷弧菌的溶血能力和利用immunoblot偵測胞內DnaK與GroEL的表現情形。
研究顯示,鹽度越低會增加創傷弧菌的死亡,而在經過低鹽適應後無論是rpoS突變株或野生株皆能對殺傷低鹽濃度產生抗性,而對高溫、酸和膽鹽則無抗性產生,但經過低鹽適應後的創傷弧菌反而會對膽鹽更為敏感。而經過低鹽適應後創傷弧菌C7184o的溶血能力有顯著的增加,但在兩株菌中並沒有觀察到DnaK和GroEL的表現有顯著的改變。根據結果顯示,創傷弧菌對低鹽壓力的適應可能與RpoS無直接的關連性,而低鹽適應的機制仍需進一步研究。
Vibrio vulnificus is a gram-negative bacterium found in estuarine and coastal waters. This bacterium is capable of causing rapidly fatal septicemia and wound infections subsequent to the ingestion of raw or undercooked seafood or following the exposure of wounds to water containing this pathogen. The rpoS-encoded sigma factor, RpoS (σs), is a central regulator in adaptation to diverse types of stresses. These stresses include starvation, osmotic stress, low pH, nonoptimal high or low temperatures, and oxidative damage.
In order to study the role of rpoS in the adaptation of V. vulnificus to hypoosmotic stress, V. vulnificus C7184o and rpoS mutant V. vulnificus AH1 were examined in this study. In addition, we also observed hemolytic ability and DnaK and GroEL expression of V. vulnificus after adaptation. Our data showed that decrease of the salinity increased the death of V. vulnificus. Adaptive hypoosmotic tolerance protected V. vulnificus against low salinity but not against the the rpoS-regulated high temperature and acid stresses. There was no notable change of expression of DnaK and GroEL in both strains but the hemolytic ability of V. vulnificus C7184o increased after adaptation. These results showed that there was no remarkable difference of the response to hypoosmotic stress between both strains and indicated that RpoS may not directly involved in the adaptation to low salinity stress.
目錄……………………………………………………………………………………i
圖表目錄………………………………………………………………………...…...iii
中文摘要……………………………………………………………………………..iv
英文摘要…………………………………………………………………………….vi
壹、緒 論……………………………………………………………………………..1
一、 創傷弧菌的特性………………………………………………………………..2
一般特性………………………………………………………………………………2
致病情形及致病因子…………………………………………………………………2
二、張壓壓力對細菌的影響…………………………………………………………4
三、RpoS與環境壓力的關係……………………………………………………….10
四、DnaK和GroEL與環境壓力的關係…………………………………………….12
貳、本研究的目的與實驗設計………………………………………………………15
參、材料與方法………………………………………………………………………16
菌株的選擇…………………………………………………………………………..16
藥品與器材…………………………………………………………………………..16
抗體…………………………………………………………………………………..17
培養基成分…………………………………………………………………………..18
菌株的保存與培養…………………………………………………………………..23
生長曲線之測定……………………………………………………………………..23
不同鹽度對創傷弧菌C7184o和AH1的殺傷力………………………………….24
創傷弧菌C7184o和AH1的低鹽適應……………………………………………..24
創傷弧菌對C7184o和AH1環境壓力敏感性分析………………………………..25
低鹽適應下對環境壓力敏感性分析………………………………………………..25
1.低鹽的敏感性……………………………………………………………………...25
2.酸的敏感性 ………………………………………………………………………….26
3.高溫的敏感性……………………………………………………………………...26
4.膽鹽的敏感性……………………………………………………………………...26
chloramphenicol對低鹽適應的影響………………………………………………...26
溶血能力的檢驗……………………………………………………………………….27
創傷弧菌整體蛋白質萃取和定量…………………………………………………..28
SDS-PAGE蛋白質電泳 ……………………………………………………………….28
DnaK與GroEL的immunoblotting……………………………………………………29
統計方法……………………………………………………………………………….30
肆、結果………………………………………………………………………………31
創傷弧菌C7184o和AH1生長曲線……………………………………………….31
不同低鹽鹽度對創傷弧菌C7184o和AH1的殺傷情形…………………………..31
創傷弧菌C7184o和AH1對環境壓力的敏感性…………………………………...31
創傷弧菌低鹽適應分析……………………………………………………………..32
Chloramphenicol對低鹽適應的影響………………………………………………….34
創傷弧菌C7184o和AH1的溶血能力……………………………………………...35
創傷弧菌經低鹽適應後GroEL和DnaK的表現情形…………………………..35
伍、討論………………………………………………………………………………37
創傷弧菌對低鹽的反應與耐受性…………………………………………………..37
RpoS對於低鹽的適應與其他壓力的關係………………………………………...38
創傷弧菌經低鹽適應後對其他環境壓力的敏感性………………………………..39
創傷弧菌低鹽適應與毒性的變化…………………………………………………..42
創傷弧菌在低鹽適應時DnaK和GroEL的表現情形…………………………..43
創傷弧菌低鹽適應可能的機制……………………………………………………..44
陸、參考文獻…………………………………………………………………………47
表一、創傷弧菌經低鹽適應後其他環境壓力敏感性測試的壓力條件……………57
表二、創傷弧菌C7184o和AH1溶血titer的檢驗………………………………….58
圖一、創傷弧菌C7184o和AH1的生長曲線………………………………………59
圖二、不同鹽度對創傷弧菌C7184o和AH1的殺傷力…………………………….60
圖三、創傷弧菌C7184o指數期的菌體在低鹽適應後對低鹽的敏感性………….61
圖四、創傷弧菌AH1指數期的菌體在低鹽適應後對低鹽的敏感性……………...62
圖五、創傷弧菌C7184o和AH1指數期的菌體在經過低鹽適應後對低鹽的敏感性……………………………………………………………………………………..63
圖六、創傷弧菌C7184o穩定期的菌體在低鹽適應後對低鹽的敏感性………….64
圖七、創傷弧菌AH1穩定期的菌體在低鹽適應後對低鹽的敏感性……………...65
圖八、創傷弧菌C7184o指數期的菌體在低鹽適應後對45℃的敏感性…………66
圖九、創傷弧菌AH1指數期的菌體在低鹽適應後對45℃的敏感性……………..67
圖十、創傷弧菌C7184o指數期的菌體在低鹽適應後對酸PH3.5的敏感性…….68
圖十一、創傷弧菌AH1指數期的菌體在低鹽適應後對酸PH3.5的敏感性……..69
圖十二、創傷弧菌C7184o指數期的菌體在低鹽適應後對5%膽鹽的敏感性…..70
圖十三、創傷弧菌AH1指數期的菌體在低鹽適應後對5%膽鹽的敏感性……….71
圖十四、創傷弧菌C7184o指數期的菌體在含有chloramphenicol的低鹽環境適應後對低鹽的敏感性…………………………………………………………………….72
圖十五、創傷弧菌C7184o經過0.4% NaCl適應60分鐘後GroEL表現情形…..73
圖十六、創傷弧菌AH1經過0.4% NaCl適應60分鐘後GroEL的表現情形…...74
圖十七、創傷弧菌C7184o經過0.4% NaCl適應60分鐘後DnaK 的表現情形….75
圖十八、創傷弧菌AH1經過0.4% NaCl適應60分鐘後DnaK的表現情形…………76
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